ABSTRACT Reflecting a particular physiological state, biomarkers are very important for earlier diagnosis of diseases, realizing personalized medicine, as well as facilitating drug development and discovery. For these reasons, significant research efforts are currently being directed towards biomarker discovery and transitioning them into clinical use. AM Biotech is developing a new discovery proteomics platform called ?Unified Process for Discovering Protein Biomarkers and their X-Aptamers? (Unified Process). This technology is a unique proteomics tool created from the combination of AM Biotech's bead-based X-Aptamer selection platform, multi- color flow cytometry, and liquid chromatography tandem mass spectrometry. This Unified Process is highly attractive in that it allows for multiplexing, detection of low abundance differences and relatively quick and efficient processing. However, most importantly, it integrates the major stages of the protein biomarker pipeline: 1) discovery (sometimes separated into candidate identification and prioritization), 2) verification, and 3) clinical validation into a more efficient pipeline. Unlike typical protein biomarker discovery techniques such as ?shotgun? proteomics and 1D/2D gels coupled with MS, which includes difference gel electrophoresis (DIGE), the Unified Process identifies an X-Aptamer (XA) affinity reagent for every candidate biomarker. The availability of an affinity reagent to each candidate biomarker from the earliest stage of discovery is transformative to the pipeline because there are no assays for the vast majority of human proteins and such assays must be developed de novo. The XAs, selected against native proteins in their native environment enable multiplexed downstream assays, protein concentration from samples for more precise identification of the candidates using MS, and the ability to quickly develop an affinity-enrichment multiple reaction monitoring MS assay (AEMRM MS). The Unified Process consists of four key steps, and of these, the most important of which is the initial flow cytometry analysis of beads bound to differentially labeled proteomes. To date, AM Biotech has established flow cytometry conditions that will allow differences between two proteomes labeled with different fluorophores to be analyzed. In this application, an optimized 4-color process will be developed. This will require several inter-related variables to be studied. Once conditions for 4-color sorting are established, spiking experiments will be used to characterize the performance of the process. Successful completion of the specific aims will pave the way for a Phase II investigation to integrate, optimize and fully characterize the remaining steps of the Unified Process and to use the process to analyze patient plasma samples.